Structural Elucidation and Antiviral Activity of Covalent Cathepsin L Inhibitors

Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.


Contents
Mass change of 477.7 Da for pH of 4.0 and 493.8 Da for pH of 6.0 between main peak of cathepsin L incubated with or without CAA0225 corresponds well with the molecular weight of the inhibitor 487.55 Da (±10 Da).Notably, the mass difference between the first and the second peak of CatL is 129 Da, which corresponds well to the mass of a glutamic acid residue (129.12Da).This would indicate that cleavage of the pro-peptide was either at the amino acid position 113 or 114 for the respective species.S9 **no interaction with SARS-CoV-2 M pro according to nanoDSF (Figure S3)

Figure S1 .
Figure S1.General schematic reaction of an aldehyde (A), a ketone (B) or succinyl epoxide (C) with the catalytic thiol of CatL.The reactions in (A) and (B) are reversible.

Figure S2 .
Figure S2.Dynamic light scattering indicating monomeric CatL in solution used for in vitro experiments with a hydrodynamic radius Rh of 2.97 nm.The scattering data was accumulated over 100 s to obtain the auto-correlation function (ACF) shown in (A).The particle radius distribution shown in (B) was obtained using the Stokes-Einstein equation, for which the diffusion constant was determined via the CONTIN algorithm.

Figure S3 .Figure S4 .
Figure S3.NanoDSF assay.Comparison of thermal stability of SARS-CoV-2 M pro as a relative measure for compound affinity.The melting temperature difference ΔTm in the presence of a compound (relative to apo M pro in the absence of DMSO) is provided for a 2:1 compound to protein mixing ratio and evaluated based on a one-site binding fit using the EMBL online data-analysis platform eSPC.The melting temperature determined for SARS-CoV-2 M pro is Tm = 328.2± 0.2 K.As an additional negative control, bovine serum albumin in the absence of a compound (Tm = 333.2± 0.4 K) and in the presence of CI-XII (Tm = 333.4± 0.8 K) at a 2:1 compound to protein mixing ratio were compared.

Figure S5 .
Figure S5.(A) Binding site and superposition of covalently bound BOCA (N-BOC-2-aminoacetaldehyde) from the four individual CatL molecules found in the ASU fading from green to yellow (PDB 8B4F).(B) Two-dimensional interaction plot based on chain A according to Discovery Studio.The BOC group is located in the S2 subsite.

Figure S6 .
Figure S6.MALDI-TOF analysis of cathepsin L with and without covalently bound inhibitor CAA0225.Activated matured cathepsin L (3 µM) was incubated with inhibitor CAA0225 at pH of 4.0 and 6.0 in a molar ratio 1:10 for 15 min at 37 °C and subjected to MALDI-TOF analysis.Mass change of 477.7 Da for pH of 4.0 and 493.8 Da for pH of 6.0 between main peak of cathepsin L incubated with or without CAA0225 corresponds well with the molecular weight of the inhibitor 487.55 Da (±10 Da).Notably, the mass difference between the first and the second peak of CatL is 129 Da, which corresponds well to the mass of a glutamic acid residue (129.12Da).This would indicate that cleavage of the pro-peptide was either at the amino acid position 113 or 114 for the respective species.

Figure S7 .
Figure S7.Inactivation of CatL.In vitro inhibition assay comparing E-64, CLIK148 and CAA0225 at pH 6 and pH 4. The inactivation rates (yaxis; per second) are shown for each inhibitor concentration (x-axis; log nM).CAA0225 is shown in purple, E-64 in green and CLIK148 in red.Datapoints, obtained at pH 6 and 4 are shown as circles and triangles, respectively.The standard error boxes for each datapoint are shown.Considering an acidic pH value of the lysosomal environment, inhibition of CatL by CAA0225 and CLIK148 is stronger at pH 6 than at pH 4. This is in contrast to E-64, which is a stronger inhibitor at the lower pH of 4. Assuming a pKa value around 4 for the carboxylate group of E-64, a larger fraction is deprotonated in an environment at pH 6.Thus, the higher negative charge of this substituent at pH 6 weakens the electrophilicity of the epoxide carbon and hence reducing the affinity to the reactive cysteine.

Figure S8 .
Figure S8.Compound and active site superimposition shows conserved hydrogen bonding with the main chain atoms of amino acids Gln19, Gly68 and Asp162 of CatL and indicates ways to complement individual moieties in different enzyme subsites.(A) MG-132 (yellow), CI-III (green) and CI-XII (light blue) as well as (B) the epoxides CAA0225 (purple), CLIK148 (brown), E-64 (cyan) and E-64d (magenta) binding to CatL as individually shown in figures 3-6.(C) Superposition of CatL (PDB 8AHV), CatB (PDB 2IPP) and CatK (PDB 5TUN) showing widely conserved hydrogen bonding.Dashed lines indicate hydrogen bonds of CI-XII in panel A and CAA0225 in panel B. Hydrogen bonds of both these compounds with CatL (green) are shown in panel C. Compounds are shown in stick representation, surrounding amino acid residues as lines (chain A of the ASU).Notably, a hydrogen bond with His163 is observed only for the ketoamides CI-XII and 13b and a hydrogen bond with the side chain of Asp162, indicated to be related to enzyme specificity, is exclusively formed by CAA0225.(D) Alignment of residues surrounding the active site cysteine of calpain (PDB 1ZCM) and CatL (PDB 7Z3T) indicating some conservation of interacting residues and underlining the potential to inhibit CatL by active site calpain inhibitors.Despite the potent inhibition of CatL by the proteasome inhibitor MG-132 and a few coronavirus M pro inhibitors, the structural similarity is lower and the active site alignment of these enzymes was not included.The interaction of calpeptin with human CatL and SARS-CoV-2 M pro can structurally be compared based on Reinke et al. 1

Table S1 .
Chemical and kinetic properties of CatL inhibitors.Page S9

Table S4 .
Compound supply and quality parameters.Page S11

Table S7 . Crystallographic table, data processing and refinement (3/3).
*Values in parentheses refer to the outer resolution shell.